The phosphoramidate modification of oligonucleotides is long known (Vorob'ev, O. E., et al., Doklady Akademii Nauk SSSR 166(1) (1966) 95-98). The frequent used synthesis is based on the strategy of converting an H-Phosphonate with CCl4 in the presence of an nucleophilic amine to the corresponding phosphoramidate (Froehler, B. C., Tetrahedron Letters 27(46) (1986) 5575-5578).
This strategy is also used for building up non-nucleosidic oligophosphoramidates with amine residues at the P atom (WO 95/23160=EP 0 751 948) and Fathi, R., et al., J. Org. Chem. 61 (1996) 5600-5609) which are modified in different manners. The residues are introduced during an oxidation step of a H-phosphonate. Only nucleophilic amines could be used by using this synthesis strategy. This results in a nitrogen atom which is protonated under acidic conditions which results in hydrolysis. Stereoselective synthesis is not disclosed in this context.
Acceptor-substituted amines could not be used since they are not nucleophilic and will not react with the dichlorophosphonate intermediate.
The P—N bond in standard phosphoramidates is known to be labile (Tomasz, J., and Ludwig, J., Nucleosides & Nucleotides 3(1) (1984) 45-60), especially under slightly acid conditions.
Baschang, G., and Kvita, V., Angew. Chem. 85(1) (1973) 43-44 describe the reaction of a nucleotide phosphoric acid triester with azides such as methylsulfonyl azide to prepare tri-alkyl(aryl)imidophosphates which are, however, unstable and decompose.
Nielsen, J., and Caruthers, M. H., J. Am. Chem. Soc. 110 (1988) 6275-6276 describe the reaction of deoxynucleoside phosphites provided with a 2-cyano-1,1-dimethylethyl protective group in the presence of alkyl azide. Furthermore, the authors suggest that this principle is suitable for preparing nucleotides that are modified on the phosphate residue without elucidating which types of modifications prepared with the aid of the disclosed method could have particular advantages. In particular the authors suggest the introduction of alkyl residues.
Therefore it was one object of the invention to provide compounds with a stabilized phosphoamidate linkage.
It was a further object of the invention to provide a new and simple preparative approach to generate a plurality of different individual oligomers consisting of a defined sequence of selected monomeric units, whereas the stereochemistry of the monomeric units is defined.
The monomeric units themselves are build of two parts, a phosphoramidate containing part and a spacer part. The monomeric units themselves contain two groups R1 and R2 where R1 is connected to the phosphoramidate containing part and R2 is connected to the spacer part of the monomeric unit.